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High-intensity femtosecond pulses from an X-ray free-electron laser enable pump–probe experiments for the investigation of electronic and nuclear changes during light-induced reactions. On timescales ranging from femtoseconds to milliseconds and for a variety of biological systems, time-resolved serial femtosecond crystallography (TR-SFX) has provided detailed structural data for light-induced isomerization, breakage or formation of chemical bonds and electron transfer 1 , 2 . However, all ultrafast TR-SFX studies to date have employed such high pump laser energies that nominally several photons were absorbed per chromophore 3 – 17 . As multiphoton absorption may force the protein response into non-physiological pathways, it is of great concern 18 , 19 whether this experimental approach 20 allows valid conclusions to be drawn vis-à-vis biologically relevant single-photon-induced reactions 18 , 19 . Here we describe ultrafast pump–probe SFX experiments on the photodissociation of carboxymyoglobin, showing that different pump laser fluences yield markedly different results. In particular, the dynamics of structural changes and observed indicators of the mechanistically important coherent oscillations of the Fe–CO bond distance (predicted by recent quantum wavepacket dynamics 21 ) are seen to depend strongly on pump laser energy, in line with quantum chemical analysis. Our results confirm both the feasibility and necessity of performing ultrafast TR-SFX pump–probe experiments in the linear photoexcitation regime. We consider this to be a starting point for reassessing both the design and the interpretation of ultrafast TR-SFX pump–probe experiments 20 such that mechanistically relevant insight emerges. Ultrafast time-resolved serial femtosecond crystallography is used to investigate a photodissociation reaction in a protein, revealing the strong impact of the pump laser fluence on the structural changes  and the reaction mechanism.

Replacing the iron atom in Fe-porphyrin IX proteins with a noble-metal atom enables the creation of enzymes that catalyse reactions not catalysed by native Fe-enzymes or other metalloenzymes; this approach could be used to generate other artificial enzymes that could catalyse a wide range of abiological transformations. Changing the 'metallo' in metalloenzymes Naturally occurring metalloenzymes are promising alternatives to transition-metal catalysts and non-metal enzymes for the synthesis of chemicals and biologically active compounds, but they catalyse only a narrow range of reactions. One way of broadening that range is to replace the native catalytic metal with an abiological metal. John Hartwig and colleagues report the effect of substituting the iron atom in Fe-porphyrin IX (Fe-PIX) proteins. Myoglobin variants containing an Ir(Me) site catalyse the functionalization of C–H bonds to form C–C bonds and add carbenes to β-substituted vinylarenes and unactivated aliphatic α-olefins. Directed evolution of the Ir(Me)-myoglobin generates mutants that form either enantiomer of the products of C–H insertion and catalyse the enantio- and diastereoselective cyclopropanation of unactivated olefins. The rich chemistry of free metalloporphyrins and the ease of preparation and evolution of substituted haem proteins using the methods adopted here open the way to the creation of many artificial metalloenzymes. Enzymes that contain metal ions—that is, metalloenzymes—possess the reactivity of a transition metal centre and the potential of molecular evolution to modulate the reactivity and substrate-selectivity of the system 1 . By exploiting substrate promiscuity and protein engineering, the scope of reactions catalysed by native metalloenzymes has been expanded recently to include abiological transformations 2 , 3 . However, this strategy is limited by the inherent reactivity of metal centres in native metalloenzymes. To overcome this limitation, artificial metalloproteins have been created by incorporating complete, noble-metal complexes within proteins lacking native metal sites 1 , 4 , 5 . The interactions of the substrate with the protein in these systems are, however, distinct from those with the native protein because the metal complex occupies the substrate binding site. At the intersection of these approaches lies a third strategy, in which the native metal of a metalloenzyme is replaced with an abiological metal with reactivity different from that of the metal in a native protein 6 , 7 , 8 . This strategy could create artificial enzymes for abiological catalysis within the natural substrate binding site of an enzyme that can be subjected to directed evolution. Here we report the formal replacement of iron in Fe-porphyrin IX (Fe-PIX) proteins with abiological, noble metals to create enzymes that catalyse reactions not catalysed by native Fe-enzymes or other metalloenzymes 9 , 10 . In particular, we prepared modified myoglobins containing an Ir(Me) site that catalyse the functionalization of C–H bonds to form C–C bonds by carbene insertion and add carbenes to both β-substituted vinylarenes and unactivated aliphatic α-olefins. We conducted directed evolution of the Ir(Me)-myoglobin and generated mutants that form either enantiomer of the products of C–H insertion and catalyse the enantio- and diastereoselective cyclopropanation of unactivated olefins. The presented method of preparing artificial haem proteins containing abiological metal porphyrins sets the stage for the generation of artificial enzymes from innumerable combinations of PIX-protein scaffolds and unnatural metal cofactors to catalyse a wide range of abiological transformations.

Background Myoglobin clearance in acute kidney injury requiring renal replacement therapy is important because myoglobin has direct renal toxic effects. Clinical data comparing different modalities of renal replacement therapy addressing myoglobin clearance are limited. This study aimed to compare two renal replacement modalities regarding myoglobin clearance. Methods In this prospective, randomized, single-blinded, single-center trial, 70 critically ill patients requiring renal replacement therapy were randomized 1:1 into an intervention arm using continuous veno-venous hemodialysis with high cutoff dialyzer and a control arm using continuous veno-venous hemodiafiltration postdilution with high-flux dialyzer. Regional citrate anticoagulation was used in both groups to maintain the extracorporeal circuit. The concentrations of myoglobin, urea, creatinine, β2-microglobulin, interleukin-6 and albumin were measured before and after the dialyzer at 1 h, 6 h, 12 h, 24 h and 48 h after initiating continuous renal replacement therapy. Results Thirty-three patients were allocated to the control arm (CVVHDF with high-flux dialyzer) and 35 patients to the intervention arm (CVVHD with high cutoff dialyzer). Myoglobin clearance, as a primary endpoint, was significantly better in the intervention arm than in the control arm throughout the whole study period. The clearance values for urea and creatinine were higher in the control arm. There was no measurable albumin clearance in both arms. The clearance data for β 2 -microglobulin and interleukin-6 were non-inferior in the intervention arm compared to those for the control arm. Dialyzer lifespan was 57.0 [38.0, 72.0] hours in the control arm and 70.0 [56.75, 72.0] hours in the intervention arm ( p  = 0.029). Conclusions Myoglobin clearance using continuous veno-venous hemodialysis with high cutoff dialyzer and regional citrate anticoagulation is better than that with continuous veno-venous hemodiafiltration with regional citrate anticoagulation. Trial registration German Clinical Trials Registry (DRKS00012407); date of registration 23/05/2017. https://www.drks.de/drks_web/navigate.do?navigationId=trial.HTML&TRIAL_ID=DRKS00012407 .

Hemoglobin and myoglobin are widely responsible for oxygen transport and storage (see the Perspective by Rezende ). The ability of diving mammals to obtain enough oxygen to support extended dives and foraging is largely dependent on muscle myoglobin (Mb) content. Mirceta et al. (p. 1234192 ) found that in mammalian lineages with an aquatic or semiaquatic lifestyle, Mb net charge increases, which may represent an adaptation to inhibit self-association of Mb at high intracellular concentrations. Epistasis results from nonadditive genetic interactions and can affect phenotypic evolution. Natarajan et al. (p. 1324 ) found that epistatic interactions were able to explain the increased hemoglobin oxygen-binding affinity observed in deer mice populations at high altitude. In mammals, the offloading of oxygen from hemoglobin is facilitated by a reduction in the blood's pH, driven by metabolically produced CO 2 . However, in fish, a reduction in blood pH reduces oxygen carrying capacity of hemoglobin. Rummer et al. (p. 1327 ) implanted fiber optic oxygen sensors within the muscles of rainbow trout and found that elevated CO 2 levels in the water led to acidosis and elevated oxygen tensions. Increasing the number of charged amino acids allows for higher myoglobin concentrations in the muscles of diving mammals. [Also see Perspective by Rezende ] Extended breath-hold endurance enables the exploitation of the aquatic niche by numerous mammalian lineages and is accomplished by elevated body oxygen stores and adaptations that promote their economical use. However, little is known regarding the molecular and evolutionary underpinnings of the high muscle myoglobin concentration phenotype of divers. We used ancestral sequence reconstruction to trace the evolution of this oxygen-storing protein across a 130-species mammalian phylogeny and reveal an adaptive molecular signature of elevated myoglobin net surface charge in diving species that is mechanistically linked with maximal myoglobin concentration. This observation provides insights into the tempo and routes to enhanced dive capacity evolution within the ancestors of each major mammalian aquatic lineage and infers amphibious ancestries of echidnas, moles, hyraxes, and elephants, offering a fresh perspective on the evolution of this iconic respiratory pigment.

Directed evolution is a powerful tool for improving existing properties and imparting completely new functionalities to proteins 1 – 4 . Nonetheless, its potential in even small proteins is inherently limited by the astronomical number of possible amino acid sequences. Sampling the complete sequence space of a 100-residue protein would require testing of 20 100 combinations, which is beyond any existing experimental approach. In practice, selective modification of relatively few residues is sufficient for efficient improvement, functional enhancement and repurposing of existing proteins 5 . Moreover, computational methods have been developed to predict the locations and, in certain cases, identities of potentially productive mutations 6 – 9 . Importantly, all current approaches for prediction of hot spots and productive mutations rely heavily on structural information and/or bioinformatics, which is not always available for proteins of interest. Moreover, they offer a limited ability to identify beneficial mutations far from the active site, even though such changes may markedly improve the catalytic properties of an enzyme 10 . Machine learning methods have recently showed promise in predicting productive mutations 11 , but they frequently require large, high-quality training datasets, which are difficult to obtain in directed evolution experiments. Here we show that mutagenic hot spots in enzymes can be identified using NMR spectroscopy. In a proof-of-concept study, we converted myoglobin, a non-enzymatic oxygen storage protein, into a highly efficient Kemp eliminase using only three mutations. The observed levels of catalytic efficiency exceed those of proteins designed using current approaches and are similar with those of natural enzymes for the reactions that they are evolved to catalyse. Given the simplicity of this experimental approach, which requires no a priori structural or bioinformatic knowledge, we expect it to be widely applicable and to enable the full potential of directed enzyme evolution. NMR spectroscopy has been used to guide the directed evolution of myoglobin to a Kemp eliminase with high catalytic efficiency, outlining an approach that is likely to be generally applicable to other enzyme activities.

The speciose mammalian order Eulipotyphla (moles, shrews, hedgehogs, solenodons) combines an unusual diversity of semi-aquatic, semi-fossorial, and fossorial forms that arose from terrestrial forbearers. However, our understanding of the ecomorphological pathways leading to these lifestyles has been confounded by a fragmentary fossil record, unresolved phylogenetic relationships, and potential morphological convergence, calling for novel approaches. The net surface charge of the oxygen-storing muscle protein myoglobin (Z Mb ), which can be readily determined from its primary structure, provides an objective target to address this question due to mechanistic linkages with myoglobin concentration. Here, we generate a comprehensive 71 species molecular phylogeny that resolves previously intractable intra-family relationships and then ancestrally reconstruct Z Mb evolution to identify ancient lifestyle transitions based on protein sequence alone. Our phylogenetically informed analyses confidently resolve fossorial habits having evolved twice in talpid moles and reveal five independent secondary aquatic transitions in the order housing the world’s smallest endothermic divers. The shrews, moles and hedgehogs that surround us all belong to the same large group of insect-eating mammals. While most members in this ‘Eulipotyphla order’ trot on land, some, like moles, have evolved to hunt their prey underground. A few species, such as the water shrews, have even ventured to adopt a semi-aquatic lifestyle, diving into ponds and streams to retrieve insects. These underwater foragers share unique challenges, burning a lot of energy and losing heat at a high rate while not being able to store much oxygen. It is still unclear how these semi-aquatic habits have come to be: the fossil record is fragmented and several species tend to display the same adaptations even though they have evolved separately. This makes it difficult to identify when and how many times the Eulipotyphla species started to inhabit water. The protein myoglobin, which gives muscles their red color, could help in this effort. This molecule helps muscles to capture oxygen from blood, a necessary step for cells to obtain energy. Penguins, seals and whales, which dive to get their food, often have much higher concentration of myoglobin so they can spend extended amount of time without having to surface for air. In addition, previous work has shown that eight groups of mammalian divers carry genetic changes that help newly synthetized myoglobin proteins to not stick to each other. This means that these animals can store more of the molecule in their muscles, increasing their oxygen intake and delivery. He et al. therefore speculated that all semi-aquatic Eulipotyphla species would carry genetic changes that made their myoglobin less likely to clump together; underground species, which also benefit from absorbing more oxygen, would display intermediate alterations. In addition, reconstructing the myoglobin sequences from the ancestors of living species would help to spot when the transition to aquatic life took place. A variety of approaches were harnessed to obtain myoglobin and other sequences from 55 eulipotyphlan mammals, which then were used to construct a strongly supported family tree for this group. The myoglobin results revealed that from terrestrial to subterranean to semi-aquatic species, genetic changes took place that would diminish the ability for the proteins to stick to each other. This pattern also showed that semi-aquatic lifestyles have independently evolved five separate times – twice in moles, three times in shrews. By retracing the evolutionary history of specific myoglobin properties, He et al. shed light on how one of the largest orders of mammals has come to be fantastically diverse.

Water dispersible L-glutamic acid (Glu) functionalized cesium lead bromide perovskite quantum dots (CsPbBr 3 PQDs), namely CsPbBr 3 @Glu PQDs were synthesized and used for the fluorescence “turn-off” detection of myoglobin (Myo). The as-prepared CsPbBr 3 @Glu PQDs exhibited an exceptional photoluminescence quantum yield of 25% and displayed emission peak at 520 nm when excited at 380 nm. Interestingly, the fluorescence “turn-off” analytical approach was designed to detect Myo using CsPbBr 3 @Glu PQDs as a simple optical probe. The developed probe exhibited a wide linear range (0.1–25 µM) and a detection limit of 42.42 nM for Myo sensing. The CsPbBr 3 @Glu PQDs-based optical probe provides high ability to determine Myo in serum and plasma samples. Graphical Abstract

Protein functions require conformational motions. We show here that the dominant conformational motions are slaved by the hydration shell and the bulk solvent. The protein contributes the structure necessary for function. We formulate a model that is based on experiments, insights from the physics of glass-forming liquids, and the concepts of a hierarchically organized energy landscape. To explore the effect of external fluctuations on protein dynamics, we measure the fluctuations in the bulk solvent and the hydration shell with broadband dielectric spectroscopy and compare them with internal fluctuations measured with the Mössbauer effect and neutron scattering. The result is clear. Large-scale protein motions are slaved to the fluctuations in the bulk solvent. They are controlled by the solvent viscosity, and are absent in a solid environment. Internal protein motions are slaved to the beta fluctuations of the hydration shell, are controlled by hydration, and are absent in a dehydrated protein. The model quantitatively predicts the rapid increase of the mean-square displacement above [almost equal to]200 K, shows that the external beta fluctuations determine the temperature- and time-dependence of the passage of carbon monoxide through myoglobin, and explains the nonexponential time dependence of the protein relaxation after photodissociation.

Myoglobin (Mb), an important cardiac marker, plays a crucial role in diagnosing, monitoring, and evaluating the condition of patients with cardiovascular diseases. Here, we propose a label-free photoelectrochemical (PEC) sensor for the detection of Mb through target regulated the photoactivity of Ag.sub.2S/FeOOH heterojunction. The Ag.sub.2S/FeOOH nanospindles were synthesized and served as a sensing platform for the fabrication of bio-recognized process for Mb. Mb-aptamer was used as the responsive group to grasp the target Mb in a real sample due to its advantages of strong affinity, high stability, and ease of preparation. Mb-aptamer immunocomplex is formed in the presence of Mb, which hinders the interfacial electron transfer and then reduce the photocurrent. The proposed PEC aptasensor exhibited excellent analytical performance including wide linear range (1.0 pg mL.sup.-1 50 ng mL.sup.-1), low limit of detection (0.28 pg mL.sup.-1), and good selectivity and stability. This work introduces an innovative approach to PEC aptasensor, offering a promising method for precise determination of human biomarkers. Graphical

The nitrite anion is reduced to nitric oxide (NO{bullet}) as oxygen tension decreases. Whereas this pathway modulates hypoxic NO{bullet} signaling and mitochondrial respiration and limits myocardial infarction in mammalian species, the pathways to nitrite bioactivation remain uncertain. Studies suggest that hemoglobin and myoglobin may subserve a fundamental physiological function as hypoxia dependent nitrite reductases. Using myoglobin wild-type (⁺/⁺) and knockout (⁻/⁻) mice, we here test the central role of myoglobin as a functional nitrite reductase that regulates hypoxic NO{bullet} generation, controls cellular respiration, and therefore confirms a cytoprotective response to cardiac ischemia-reperfusion (I/R) injury. We find that myoglobin is responsible for nitrite-dependent NO{bullet} generation and cardiomyocyte protein iron-nitrosylation. Nitrite reduction to NO{bullet} by myoglobin dynamically inhibits cellular respiration and limits reactive oxygen species generation and mitochondrial enzyme oxidative inactivation after I/R injury. In isolated myoglobin⁺/⁺ but not in myoglobin⁻/⁻ hearts, nitrite treatment resulted in an improved recovery of postischemic left ventricular developed pressure of 29%. In vivo administration of nitrite reduced myocardial infarction by 61% in myoglobin⁺/⁺ mice, whereas in myoglobin⁻/⁻ mice nitrite had no protective effects. These data support an emerging paradigm that myoglobin and the heme globin family subserve a critical function as an intrinsic nitrite reductase that regulates responses to cellular hypoxia and reoxygenation. myoglobin knockout mice